Mutational studies uncover non-native structure in the dimeric kinetic intermediate of the H2A-H2B heterodimer

Matthew R Stump; Lisa M Gloss

doi:10.1016/j.jmb.2010.06.034

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Mutational studies uncover non-native structure in the dimeric kinetic intermediate of the H2A-H2B heterodimer

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Mutational studies uncover non-native structure in the dimeric kinetic intermediate of the H2A-H2B heterodimer

Matthew R Stump and Lisa M Gloss

Journal of molecular biology, Vol.401(3), pp.518-531

08/20/2010

DOI: https://doi.org/10.1016/j.jmb.2010.06.034

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https://hdl.handle.net/2376/106357

PMCID: PMC2914809

PMID: 20600120

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Abstract

Urea

Histones - genetics

Histones - chemistry

Protein Multimerization

Protein Denaturation

Kinetics

Mutation

Protein Stability

Protein Folding

The folding pathway of the histone H2A-H2B heterodimer minimally includes an on-pathway, dimeric, burst-phase intermediate, I(2). The partially folded H2A and H2B monomers populated at equilibrium were characterized as potential monomeric kinetic intermediates. Folding kinetics were compared for initiation from isolated, folded monomers and the heterodimer unfolded in 4 M urea. The observed rates were virtually identical above 0.4 M urea, exhibiting a log-linear relationship on the final denaturant concentration. Below approximately 0.4 M urea (concentrations inaccessible from the 4-M urea unfolded state), a rollover in the rates was observed; this suggests that a component of the I(2) ensemble contains non-native structure that rearranges/isomerizes to a more native-like species. The contribution of helix propensity to the stability of the I(2) ensemble was assessed with a set of H2A-H2B mutants containing Ala and Gly replacements at nine sites, focusing mainly on the long, central alpha2 helix. Equilibrium and kinetic folding/unfolding data were collected to determine the effects of the mutations on the stability of I(2) and the transition state between I(2) and N(2). This limited mutational study indicated that residues in the alpha2 helices of H2A and H2B as well as alpha1 of H2B and both the C-terminus of alpha3 and the short alphaC helix of H2A contribute to the stability of the I(2) burst-phase species. Interestingly, at least eight of the nine targeted residues stabilize I(2) by interactions that are non-native to some extent. Given that destabilizing I(2) and these non-native interactions does not accelerate folding, it is concluded that the native and non-native structures present in the I(2) ensemble enable efficient folding of H2A-H2B.

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Title: Mutational studies uncover non-native structure in the dimeric kinetic intermediate of the H2A-H2B heterodimer
Creators: Matthew R Stump - School of Molecular Biosciences, Washington State University, Pullman, WA 99164-7520, USA
Lisa M Gloss
Publication Details: Journal of molecular biology, Vol.401(3), pp.518-531
Academic Unit: Graduate School
Publisher: England
Grant note: T32 GM008336 / NIGMS NIH HHS GM08336 / NIGMS NIH HHS R01 GM073787 / NIGMS NIH HHS R01 GM073787-04 / NIGMS NIH HHS GM073787 / NIGMS NIH HHS
Identifiers: 99900546704101842
Language: English
Resource Type: Journal article